Revealing the quantum nature of the voltage-induced conductance changes in oxygen engineered yttrium oxide-based RRAM devices.

In this work, the quasi-analog to discrete transition occurring in the current-voltage characteristic of oxygen engineered yttrium oxide-based resistive random-access memory (RRAM) devices is investigated in detail. In particular, the focus of our research is not on the absolute conductance values of this characteristic but on the magnitude of its conductance changes occurring during the reset process of the device. It is found that the detected changes correspond to conductance values predominantly of the order of the quantum unit of conductance G0 = 2e2/h, where e is the electron charge and h the Planck constant. This feature is observed even at conductance levels far above G0, i.e. where electron transport is seemingly diffusive. It is also observed that such behavior is reproducible across devices comprising yttrium oxide layers with different oxygen concentrations and measured under different voltage sweep rates. While the oxygen deficiency affects the total number of quantized conductance states, the magnitude of the changes in conductance, close to 1 G0, is invariant to the oxygen content of the functional layer.

delta-Opioid receptors are more efficiently coupled to adenylyl cyclase than to L-type Ca(2+) channels in transfected rat pituitary cells.

Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which delta-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH(3) cells with cDNAs encoding for delta-opioid receptors. In cells transfected with a relatively low delta-receptor density of 0.55 pmol/mg of protein (GH(3)DOR), activation of delta-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca(2+) current. In contrast, activation of delta-receptors in a clone that contained a higher density of delta-receptors (2.45 pmol/mg of protein) and was also coexpressed with mu-opioid receptors (GH(3)MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca(2+) current. The purpose of the present study was to determine whether these observations resulted from differences in delta-opioid receptor density between clones or interaction between delta- and mu-opioid receptors to allow the activation of different G proteins and signaling to Ca(2+) channels. Using the delta-opioid receptor alkylating agent SUPERFIT, reduction of available delta-opioid receptors in GH(3)MORDOR cells to a density similar to that of delta-opioid receptors in the GH(3)DOR clone resulted in abolishment of coupling to Ca(2+) channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by delta-opioid receptors in GH(3)MORDOR cells, delta-opioid receptor activation in GH(3)DOR cells resulted in coupling to the identical pattern of G proteins seen in GH(3)MORDOR cells. These findings suggest that different threshold densities of delta-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that delta-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca(2+) channels.

Cloned delta-opioid receptors in GH(3) cells inhibit spontaneous Ca(2+) oscillations and prolactin release through K(IR) channel activation.

Opioid receptors can couple to K(+) and Ca(2+) channels, adenylyl cyclase, and phosphatidyl inositol turnover. Any of these actions may be important in the regulation of neurotransmitter and hormone release from excitable cells. GH(3) cells exhibit spontaneous oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) and prolactin release. Activation of cloned delta-opioid receptors stably expressed in GH(3) cells inhibits both spontaneous Ca(2+) signaling and basal prolactin release. The objective of this study was to examine a possible role for K(+) channels in these processes using the patch-clamp technique, fluorescence imaging, and a sensitive ELISA for prolactin. The selective delta receptor agonist [D-Pen(2), D-Pen(2)]enkephalin (DPDPE) inhibited [Ca(2+)](i) oscillations in GH(3) cells expressing both mu and delta receptors (GH(3)MORDOR cells) but had no effect on control GH(3) cells or cells expressing mu receptors alone (GH(3)MOR cells). The inhibition of [Ca(2+)](i) oscillations by DPDPE was unaffected by thapsigargin pretreatment, suggesting that this effect is independent of inositol 1,4,5-triphosphate-sensitive Ca(2+) stores. DPDPE caused a concentration-dependent inhibition of prolactin release from GH(3)MORDOR cells with an IC(50) of 4 nM. DPDPE increased inward K(+) current recorded from GH(3)MORDOR cells but had no significant effect on K(+) currents recorded from control GH(3) cells or GH(3)MOR cells. The mu receptor agonist morphine also had no effect on currents recorded from control cells but activated inward K(+) currents recorded from GH(3)MOR and GH(3)MORDOR cells. Somatostatin activated inward currents recorded from all three cell lines. The DPDPE-sensitive K(+) current was inwardly rectifying and was inhibited by Ba(2+) but not TEA. DPDPE had no effect on delayed rectifier-, Ca(2+)-, and voltage-activated or A-type K(+) currents, recorded from GH(3)MORDOR cells. Ba(2+) attenuated the inhibition of [Ca(2+)](i) and prolactin release by DPDPE, whereas TEA had no effect, consistent with an involvement of K(IR) channels in these actions of the opioid.

Purification of an EH domain-binding protein from rat brain that modulates the gating of the rat ether-à-go-go channel.

Mutations in the gene encoding ether-à-go-go (EAG) potassium channel impair the function of several classes of potassium currents, synaptic transmission, and learning in Drosophila. Absence of EAG abolishes the modulation of a broad group of potassium currents. EAG has been proposed to be a regulatory subunit of different potassium channels. To further explore this regulatory role we searched for signaling molecules that associate with EAG protein. We have purified a approximately 95-kDa protein from rat brain membranes that binds to EAG. When co-expressed in mammalian cells this protein coimmunoprecipites with EAG and alters the gating of EAG channels. Expression of this protein is regulated during neuronal differentiation. The protein is identical to the recently reported rat protein epsin, which is an EH domain-binding protein similar to the Xenopus mitotic phosphoprotein MP90. These results show that proteins of the epsin family are modulators of channel activity that may link signaling pathways, or the cell cycle, to EAG and thus to various potassium channel functions.

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells.

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.

Functional analysis of cloned opioid receptors in transfected cell lines.

Opioids modulate numerous central and peripheral processes including pain perception neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (mu, delta, kappa). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein alpha subunits and/or beta gamma subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K+ channels and reduces adenylyl cyclase and Ca2+ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca2+ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca2+ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH3 cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.

Voltage-dependent inhibition of Ca2+ channels in GH3 cells by cloned mu- and delta-opioid receptors.

To study cloned opioid receptor binding and modulation of both adenylyl cyclase and ion channel activity, we stably expressed mu- and delta-opioid receptors in the rodent pituitary-derived GH3 cell line. GH3 cells express G proteins and voltage-activated Ca2+ channels (predominantly of the L-type). Activation of cloned rat mu-opioid receptors expressed in GH3 cells (termed GH3MOR cells) inhibits L-type Ca2+ channel activity. GH3MOR cells, further transfected with mouse delta receptor cDNA (termed GH3MORDOR cells), bound both [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE). These opioid ligands inhibited adenylyl cyclase activity (IC50 = 174 and 0.53 nM, respectively). This action of DAMGO and DPDPE was attenuated selectively by mu- and delta-opioid receptor-specific antagonists. Activation of both opioid receptors also led to inhibition of Ca2+ channel activity, measured with Ba2+ as the charge carrier using the whole-cell patch-clamp technique. Both DAMGO (1 microM) and DPDPE (1 microM) reversibly inhibited Ba2+ currents (by 17.0 +/- 1.4% and 20.7 +/- 1.3%, respectively) in GH3MORDOR cells. The inhibitory action of DPDPE was dose dependent (IC50 = 1.6 nM) and was attenuated by pretreatment with pertussis toxin (200 ng/ml) or by the inclusion of guanosine-5'-O-(2-thio)diphosphate (2 mM) in the recording electrode. Ba2+ current inhibitions by both DAMGO and DPDPE were completely reversed by depolarizing (to > 50 mV) prepulses in GH3MORDOR cells. In summary, cloned mu- and delta-opioid receptors expressed in GH3 cells voltage-dependently couple through Gi/G(o) proteins to L-type Ca2+ channels.

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Ca2+ channel and adenylyl cyclase modulation by cloned mu-opioid receptors in GH3 cells.

Members of the three classes of opioid receptors (mu, delta, and kappa) have been cloned and characterized in unexcitable cell lines using biochemical techniques. However, an important function of these cloned receptors, their coupling to voltage-activated Ca2+ channels, remains untested. We stably transfected cloned rat mu-opioid receptor cDNAs into clonal pituitary GH3 cells. GH3 cells expressing mu-opioid receptors (GH3MOR cells) bound the receptor-specific ligands [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and morphine with high affinity (Ki = 1.0 and 7.2 nM, respectively), and these ligands also potently inhibited adenylyl cyclase activity (IC50 = 21.9 and 55.2 nM, respectively). Functional coupling of mu-opioid receptors to voltage-activated Ca2+ channels was compared with that of endogenous somatostatin (SRIF) receptors in GH3MOR cells, using the patch-clamp technique, with Ba2+ as the charge carrier. DAMGO (1 microM) and SRIF (1 microM) inhibited Ba2+ currents by 23.8 +/- 1.0% and 22.9 +/- 2.5%, respectively. DAMGO (0.1 nM to 10 microM) dose-dependently inhibited Ba2+ currents, with an IC50 of 105 nM. The mu-opioid receptor agonist morphine (1 microM) inhibited currents by 13.5 +/- 1.1% and the delta-opioid receptor-selective ligand [D-Pen2,5]-enkephalin (1 microM) caused only 3.5 +/- 2.1% inhibition. The inhibitory actions of DAMGO, morphine, and [D-Pen2,5]-enkephalin were reversed by naloxone. Ba2+ current inhibitions by DAMGO and SRIF were attenuated by pertussis toxin pretreatment. Nimodipine reduced the amplitude of Ba2+ current inhibition by DAMGO, suggesting that mu-opioid receptors couple to L-type Ca2+ channels in GH3MOR cells.